Protein S levels and the risk of venous thrombosis: results from the MEGA case-control study.

In thrombophilic families, protein S deficiency is clearly associated with venous thrombosis. We aimed to determine whether the same holds true in a population-based case-control study (n = 5317). Subjects were regarded protein S deficient when protein S levels were < 2.5th percentile of the controls. Free and total protein S deficiency was not associated with venous thrombosis: free protein S < 53 U/dL, odds ratio [OR] 0.82 (95% confidence interval [CI], 0.56-1.21) and total protein S < 68 U/dL, OR 0.90 (95% CI, 0.62-1.31). When lower cutoff values were applied, it appeared that subjects at risk of venous thrombosis could be identified at levels < 0.10th percentile of free protein S (< 33 U/dL, OR 5.4; 95% CI, 0.61-48.8). In contrast, even extremely low total protein S levels were not associated with venous thrombosis. PROS1 was sequenced in 48 subjects with free protein S level < 1st percentile (< 4 6 U/dL), and copy number variations were investigated in 2718 subjects, including all subjects with protein S (free or total) < 2.5th percentile. Mutations in PROS1 were detected in 5 patients and 5 controls reinforcing the observation that inherited protein S deficiency is rare in the general population. Protein S testing and PROS1 testing should not be considered in unselected patients with venous thrombosis.

Molecular basis of protein S deficiency in China.

Protein S (ProS) is a physiological inhibitor of coagulation with an important function in the down-regulation of thrombin generation. ProS deficiency is a major risk factor for venous thrombosis. This study enrolled 40 ProS-deficient probands to investigate the molecular basis of hereditary ProS deficiency in Chinese patients. A mutation analysis was performed by resequencing the PROS1 gene. Large deletions were identified by multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 20 different mutations, including 15 novel mutations, were identified in 21 of the 40 index probands. Small mutations were detected in 18 (45.0%) probands, and large deletions were found in 3 (7.5%) probands, leaving 19 (47.5%) patients without causative variants. To evaluate the functional consequences of 2 novel missense variants, ex vivo thrombin-generation assays, bioinformatics tools, and in vitro expression studies were employed. The p.Asn365Lys ProS variant was found to have moderately impaired secretion and reduced activated protein C cofactor activity. In contrast, the p.Pro410His mutant appeared to have severely impaired secretion but full anticoagulant activity. This study is the largest investigation of ProS deficiency in China and the first investigation of the influence of Type I ProS missense mutations on the global level of coagulation function. The p.K196E mutation, which is common in the neighboring Japanese population, was not found in our Chinese population, and null mutations were common in our Chinese population but not common in Japan. Further genetic analysis is warranted to understand the causes of ProS deficiency in patients without a genetic explanation.

Dysregulation of Protein S in COVID-19.

Coronavirus Disease 2019 (COVID-19) has been widely associated with increased thrombotic risk, with many different proposed mechanisms. One such mechanism is acquired deficiency of protein S (PS), a plasma protein that regulates coagulation and inflammatory processes, including complement activation and efferocytosis. Acquired PS deficiency is common in patients with severe viral infections and has been reported in multiple studies of COVID-19. This deficiency may be caused by consumption, degradation, or clearance of the protein, by decreased synthesis, or by binding of PS to other plasma proteins, which block its anticoagulant activity. Here, we review the functions of PS, the evidence of acquired PS deficiency in COVID-19 patients, the potential mechanisms of PS deficiency, and the evidence that those mechanisms may be occurring in COVID-19.

Generation and phenotypic analysis of protein S-deficient mice.

Protein S (PS) is an important natural anticoagulant with potentially multiple biologic functions. To investigate further the role of PS in vivo, we generated Pros(+/-) heterozygous mice. In the null (-) allele, the Pros exons 3 to 7 have been excised through conditional gene targeting. Pros(+/-) mice did not present any signs of spontaneous thrombosis and had reduced PS plasma levels and activated protein C cofactor activity in plasma coagulation and thrombin generation assays. Tissue factor pathway inhibitor cofactor activity of PS could not be demonstrated. Heterozygous Pros(+/-) mice exhibited a notable thrombotic phenotype in vivo when challenged in a tissue factor-induced thromboembolism model. No viable Pros(-/-) mice were obtained through mating of Pros(+/-) parents. Most E17.5 Pros(-/-) embryos were found dead with severe intracranial hemorrhages and most likely presented consumptive coagulopathy, as demonstrated by intravascular and interstitial fibrin deposition and an increased number of megakaryocytes in the liver, suggesting peripheral thrombocytopenia. A few E17.5 Pros(-/-) embryos had less severe phenotype, indicating that life-threatening manifestations might occur between E17.5 and the full term. Thus, similar to human phenotypes, mild heterozygous PS deficiency in mice was associated with a thrombotic phenotype, whereas total homozygous deficiency in PS was incompatible with life.

Evaluation of Pro-C global for identification of defects in protein C/S anticoagulant pathway.

BACKGROUND: Detection of protein C and S deficiency forms a major investigation in the laboratory evaluation of thrombophilia screening. It has key role in the diagnosis of protein C and S deficiency. The objective of this study is to determine the utility of ProC Global as a screening test for identifying the defects of protein C and S anticoagulant pathways. METHODS: Two Hundred patients with venous thromboembolism were studied at the Department of Haematology, Armed Forces Institute of Pathology, Rawalpindi, from October 2004 to March 2006. ProC Global test (Dade Behring Diagnostics) was performed and was followed up by protein C and S assays. ProC Global is an activated partial thromboplastin time based assay in which Protac (snake venom from Aghistroden contortrix) is used for activation of the endogenous protein C of the plasma sample. The protein C activation time in the presence of the activator was set in relation to a parallel determination of PCAT/O with addition of a buffer instead of activator reagent. The ratio PCAT: PCAT/O was transformed in normalized ratio by relating them to a calibrator. Control plasma for normal range and ProC control plasma for pathological range (Dade Behring Diagnostics) were assayed in each run for quality control. RESULTS: A total of 200 patients, 132 (66%) males and 68 (34%) females with age ranging from 1 to 68 years were studied. ProC Global was positive in 29/200 (14.5%) patients. ProC Global was found to be 86% sensitive, 94% specific and its overall efficiency turned out to be 94%. CONCLUSION: Pro-C Global can be used effectively as a screening test to detect abnormalities in protein C and S anticoagulant pathways.

Clinical characteristics, genes identification and follow-up study of a patient with central venous thrombosis from a protein S deficiency pedigree.

OBJECTIVE: To explore the clinical and prognostic features of CVT caused by PROS1 gene mutations and to provide clinical experience for new oral anticoagulants, such as rivaroxaban, in the treatment of CVT with a high risk of thrombosis. PATIENTS AND METHODS: The CVT patient's clinical symptoms were described, and the brain imaging and blood coagulation tests were performed to confirm the diagnosis of CVT. The patient's family members were recruited to receive blood coagulation tests and ultrasonic examination of lower limb vessels. Genetic analysis on the pedigree was carried out to identify the responsible gene for PS deficiency. We followed-up with this patient for 24 months to evaluate the clinical outcomes, laboratory results and imaging performances of CVT. RESULTS: The patient presented with typical CVT symptoms, including headache and epilepsy. Brain CT showed hemorrhage in the bilateral frontal lobe and left occipital lobe, while MRV demonstrated that thrombus had occurred. It was reviewed that the patient and his mother had a history of bilateral leg deep vein thrombosis. Gene tests revealed that the patient and two family members carried a heterozygous mutation of PROS1 (c.751_752delAT, p.M251Vfs*17). During 24 months of follow-up study, the patient was treated with rivaroxaban continuously and recovered well, supported by an mRS score that remained below 2. Blood coagulation tests were within normal limits, and MRV revealed partial recanalization of the cerebral venous sinus. CONCLUSIONS: The frame shift mutation in the PROS1 gene (c.751_752delAT) may greatly affect the function of protein S and lead to a severe phenotype of CVT. Rivaroxaban showed a satisfying therapeutic effect in this CVT patient with hereditary thrombophilia.

Molecular basis of protein S deficiency.

Protein S deficiency (PSD) has been the most difficult to study among the classical inherited thrombophilic factors. This is in part due to the peculiar biology of protein S (PS), which has an anticoagulant role but no enzymatic activity, and because it interacts with plasma components that function in both haemostasis and inflammation. Clinically, it also has been difficult to define and standardise valuable assays to determine PS status and implication in thrombosis. Despite these drawbacks, at present heterozygous PS deficiency is well established as an autosomal dominant trait associated with an increased risk of thrombosis from data on familial and population studies. Almost two-hundred mutations have been characterised in PROS1, and approximately 30% of them have been characterised in vitro, clarifying the mechanisms leading to PSD. Furthermore, recent studies on the presence of large deletions in PROS1 have increased the number of PSD associated to PROS1 mutations. Finally, the discovery of new functions for PS, both in the anticoagulant system as well as in the interaction with cellular components through receptor tyrosine kinases, is broadening the importance of this molecule in the context of biomedicine.

Gene analysis of six cases of congenital protein S deficiency and functional analysis of protein S mutations (A139V, C449F, R451Q, C475F, A525V and D599TfsTer13).

Congenital deficiency of protein S (PS), an anticoagulant factor, leads to venous thrombosis, with onset predominantly beginning in adolescence. In the present study, gene analysis of six unrelated Japanese families diagnosed with congenital PS deficiency identified five missense mutations in the PROS1 gene - c.757C>T (Ala139Val; A139V), c.1346 G>T (Cys449Phe; C449F), c.1352G>A (Arg451Gln; R451Q), c.1424G>T (Cys475Phe; C475F) and c.1574C>T (Ala525Val; A525V) - and one frameshift mutation, c.2135delA (Asp599ThrfsTer13; D599TfsTer13). C449F, R451Q, A525V and D599TfsTer13 are novel mutations. Results from ELISA to measure PS antigen levels in culture supernatant showed that the A139V variant was similar to wild-type, but other variants showed reductions when compared with wild-type. Results from pulse-chase analysis confirmed that the A139V variant exhibited secretion equivalent to wild-type, but for the other variants, there was no extracellular secretion, and it had nearly all been degraded inside the cell within six hours. Results from pulse-chase analysis using proteasome inhibitors also showed that intracellular degradation of mutant protein was inhibited. Activity of the A139V variant was decreased to 71% of wild-type, and the phospholipid binding capacity fell to as low as 45%. These results suggest that although the A139V variant has normal secretion, it has abnormal phospholipid binding capacity, and therefore causes type II PS deficiency, in which PS activity is decreased. It is also thought that with the other variants, misfolding due to amino acid mutations causes nearly all PS to be degraded intracellularly, therefore leading to type I PS deficiency.

Protein C and protein S deficiencies.

The protein C (PC) pathway, with its cofactor protein S (PS), is an important natural antithrombotic mechanism. Both PC and PS deficiencies have been implicated in thrombophilia. The molecular basis for hereditary PC and PS deficiencies is highly heterogeneous, with a large spectrum of mutations that have various effects on the expression of the relevant allele. A small subset of patients who are homozygous or compound heterozygous for a PC gene mutation have severe thrombotic complications at birth, whereas onset occurs later in the other cases. Patients heterozygous for a PC or PS gene abnormality may develop recurrent thrombosis during adulthood, with a probability of remaining free of thrombosis of about 50% at age 45. A PC or PS gene defect is associated with the factor V Arg 506 to Gln mutation in 10% to 30% of symptomatic patients, suggesting that clinical expression is controlled by several genes in heterozygous patients.

Acquired free protein S deficiency is associated with antiphospholipid antibodies and increased thrombin generation in patients with systemic lupus erythematosus.

In order to determine whether there is a relationship between acquired free protein S deficiency and increased thrombin generation, we performed a cross-sectional study of patients with systemic lupus erythematosus (SLE). Plasma samples were assayed for free protein S and were correlated to levels of prothrombin fragments (F1 + 2); an elevated level of F1 + 2 was used as a surrogate marker for a prothrombotic state. Assays for anticardiolipin antibodies (ACA) and lupus anticoagulant (LA) were performed on two separate blood samples taken at least 3 months apart in order to detect the presence of antiphospholipid antibodies. Of the 36 subjects, 9 had reduced free protein S levels compared to 0 of 21 controls (P = 0.01) and the mean free protein S level was significantly lower in the SLE population than in controls (0.30 +/- 0.08 U/mL versus 0.43 +/- 0.10 U/mL, P < 0.001). Of the 24 subjects with antiphospholipid antibodies, 9 had reduced free protein S levels, compared to 0 of 12 subjects without antiphospholipid antibodies (P = .01). The mean F1 + 2 level was significantly higher in study subjects with reduced free protein S levels than in those with normal free protein S levels (1.22 +/- 0.50 nmol/L versus 0.78 +/- 0.27 nmol/L, P = 0.05). This study confirms an association between antiphospholipid antibodies and reduced free protein S levels and demonstrates that patients with SLE and acquired free protein S deficiency generate more thrombin than patients with SLE and normal free protein S levels. Further studies are needed to determine whether the thrombotic diathesis associated with the presence of antiphospholipid antibodies is directly caused by the concomitant presence of acquired free protein S deficiency.

Frequency of protein S deficiency in general Japanese population.

We measured protein S (PS) activity in a large group of Japanese subjects to study the frequency of PS deficiency. The study group comprised 213 men, ages 18-58 years, and 179 women, ages 18-60 years. PS activity in the total 392 subjects was 58-135% (mean +/- 2 SD), 65-135% in the men and 54-120% in the women. The men showed significantly higher levels of PS activity than the women (p<0.001). We identified 8 subjects (4 men and 4 women) in whom PS activity was lower than the mean-2SD for men and women, respectively. Moreover, we examined the classifications of PS deficiency. The frequency of PS deficiency in this study was 2.04% (Type I: 0.51%, Type II: 1.02%, Type III: 0.51%). Based on our findings, it would appear that the frequency of Type II PS deficiency in the Japanese population is approximately 1%. The screening of PS decrease should be judged by activity.

Resistance to activated protein C: evaluation of three functional assays.

Resistance to Activated Protein C (APC) was evaluated using 3 different methods: two of them were based on the prolongation of the Activated Partial Thromboplastin Time (APTT) using 2 different APTT reagents in the presence of APC, whereas the third method was based on the prolongation of prothrombin time when APC is added. The three methods were significantly correlated. APTT-based assays were sensitive to factor XII deficiency, whereas thromboplastin-based assay was sensitive to factor VII deficiency (< 0.5 UI/ml), which surestimates the response to APC. In contrast, an increase in factor VIII (F. VIII) level is associated with a decreased response to APC, when APTT-based assays are used, whereas thromboplastin-based assay is unmodified. During pregnancy, a decreased response to APC is observed, which is not only due to the increase in F. VIII, since thromboplastin-based assay is also modified. In Protein S (PS) immuno-depleted plasma, the low response to APC is corrected by addition of free PS: the thromboplastin-based assay was the most sensitive one to PS deficiency. However, in patients with congenital PS deficiency, there was no correlation between APC-resistance and free PS level. In patients with lupus anticoagulant, discrepancies were observed between the 3 methods, but with a high frequency of low response to APC. For the 3 assays, there was a good differentiation and correlation between normal and pathological results, the thromboplastin-based assay being perhaps the most discriminating. However, 3 unrelated thrombophilic patients showed normal results using thromboplastin-based assay, although they were APC-resistant using APTT-based assays. For 2 patients, this discrepancy can be explained by high levels of F. VIII. For the last patient, an abnormal F. VIII, resistant to APC can be suspected.

Acquired free protein S deficiency in children with steroid resistant nephrosis.

Plasma and urine concentrations of protein S were measured in five children with steroid-resistant nephrotic syndrome. It was found that plasma free protein S was reduced in three out of the five patients studied. Thus, acquired free protein S deficiency does occur in children with nephrotic syndrome and is one of many factors which may place them at risk for a thromboembolic event.

Familial thrombophilia: clinical and molecular analysis of Swedish families with inherited resistance to activated protein C or protein S deficiency.

This report describes the characterization of Swedish families with inherited resistance to activated protein C (APC resistance) and/or protein S deficiency, two genetic disorders associated with functional impairment of the protein C anticoagulant pathway. The APC resistance phenotype was linked to the factor V gene locus in a kindred with independent inheritance of APC resistance and protein S deficiency. A point mutation changing Arg506 to a Gln (FV:Q506) in the factor V gene was the cause of APC resistance. In studies of 50 families with hereditary APC resistance, the FV:Q506 mutation was identified in 94% (47/50) of the families, and the thrombotic risk was found to be dependent on the factor V genotype. Moreover, 18 families with hereditary deficiency of free protein S were investigated. Type I protein S deficiency (low free and total protein S) and type III deficiency (low free but normal total protein S) coexisted in 78% (14/18) of the families, suggesting the two types to be phenotypic variants of the same genetic disorder. Deficiency of free protein S was caused by equimolar relationship between protein S and beta-chain containing isoforms of C4BP. Though protein S deficiency was a strong risk factor for thrombosis, the FV:Q506 mutation was identified as an additional genetic risk factor in 39% of the families. Thus, familial thrombophilia is a multiple gene disorder. The thrombophilic tendency associated with APC resistance or protein S deficiency was related to increased levels of prothrombin fragment 1 + 2, reflecting increased activation of the common coagulation pathway.

[Selected risk factors of thrombotic complications in patients with ulcerative colitis]. / Wybrane wskazniki zagrozenia zakrzepowego u chorych z wrzodziejacym zapaleniem jelita grubego.

We have analyzed the prothrombotic risk factors in 124 patients with ulcerative colitis (UC) as compared with control subjects with other gastrointestinal disorders. The patients were hospitalized from 1991 to 2000 in the Department of Internal Medicine. Platelets level was significantly higher (p < 0.001) in UC patients as compared with control group, and aPTT was significantly (p < 0.05) prolonged respectively in UC patients as compared with control group. In the prospective pilot study we observed the decrease of plasma antithrombin level and/or decrease of protein S in approximately 22% of UC patients. The plasma protein C activity was normal in all UC patients, whereas the mean level of protein S was significantly lower (p < 0.02) in UC patients as compared with controls. Thus our data indicate that coagulation abnormalities are potential risk factors of thromboembolic complications in UC. Plasma cholesterol and triglyceride levels were above upper limit of normal values in 38% and 18% UC patients respectively, but mean values of both parameters were not significantly different between UC and control group. Our results suggest that plasma lipid changes are not independent risk factor of vascular complication in UC. In UC patients smokers were observed 4 times less than no-smokers. However, positive correlation between smoking and rise of hematocrit (F = 4.48; p < 0.05) in UC suggests that smoking may be a risk factor of vascular complications in the disease. Thromboembolic events were found in 1.6% of UC patients. In addition, chronic coronary heart disease was accompanied approximately 6% of UC patients. Evaluation of prothrombotic risk factors and associated ischaemic heart diseases may have prognostic value in the management of UC.

[Bilateral retinal venous thrombosis after 8 years in a young patient with congenital coagulopathy]. / Tromboza venoasa retiniana bilaterala, la un interval de 8 ani, la un tânar cu coagulopatie congenitala.

The issue discusses the case of a young male patient (35 years old), with bilateral retinal vein thrombosis. The onset of the ocular disease was eight years ago, in the left eye, with decreased visual acuity and complicated with neovascular glaucoma; with an adequate treatment, anatomical and functional evolution was good. A new retinal vein thrombosis, this time in the right eye, make us to concentrate our efforts on the etiology of the disease. Thus, we found a blood hyperviscosity syndrome, caused by a congenital deficiency of protein "S" and resistance at the activated protein "C", which may produce recurrent venous thrombosis.

[Free protein S (PS) in normal pregnancy: a comparison between two analytical methods]. / Proteína S libre en embarazadas normales: comparación entre dos métodos.

BACKGROUND: Pregnancy is a physiological hypercoagulable state with an increased incidence of thromboembolic phenomena. There is an increase in the concentrations of most clotting factors, a decrease in concentration of some of the natural anticoagulants and reduced fibrinolytic activity. Changes in PS levels have also been reported. AIM: To establish referral range values of functional PS and free PS antigen, during the second (2nd T) and third trimester (3rd T) of normal gestation. PATIENTS AND METHODS: Forty one normal pregnant women were included in our study, 20 during the 2nd T (22-24 weeks) and 21 during the 3rd T (29-38 weeks). Functional PS was measured by a clot based test and free PS antigen by ELISA. RESULTS: Free PS Antigen was 65.8+/-18.3% during the 2nd T and 62.3+/-16.5% during the 3rd T. The figures for normal controls were 106+/-6.5%. Functional PS was 43.8+/-13.3 and 25.9+/-14.6% during the 2nd T and 3rd T, respectively. The figures for normal controls were 97+/-24% (p <0.001 compared with pregnant women). Free PS antigen did not change from the 2nd to the 3rd T (p=NS), however functional PS fell significantly from the 2nd to the 3rd T (p <0.001) and was significantly lower than free PS antigen in both trimesters (p <0.001). CONCLUSIONS: Pregnancy is associated to a decrease in PS. This abnormality is more pronounced for functional PS than free PS antigen and functional PS falls progressively during pregnancy. These assays should not be used to screen for PS deficiency during pregnancy because they could lead to a misdiagnosis.

Resistance to activated protein C as an additional genetic risk factor in hereditary deficiency of protein S.

Inherited resistance to activated protein C (APC), which is caused by a single point mutation in the gene for factor V, is a common risk factor for thrombosis. In this study, the prevalence of APC resistance in 18 unrelated thrombosis-prone families with inherited protein S deficiency was investigated to determine its role as additional genetic risk factor for thrombosis. In addition, a detailed evaluation of the clinical manifestations in these families was performed. Venous thrombotic events had occurred in 47% of the protein S-deficient patients (64/136) and in 7% of relatives without protein S deficiency (14/191). As estimated from Kaplan-Meier analysis, 50% of protein S-deficient family members and 12% of those without protein S deficiency had had manifestation of venous thromboembolism at the age of 45 years. The age at the first thrombotic event ranged from 10 to 81 years (mean, 32.5 years) and a large intrafamilial and interfamilial variability in expression of thrombotic symptoms was seen. The factor V gene mutation related to APC resistance was present in 6 (38%) of 16 probands available for testing; in total, the mutation was found in 7 (39%) of the 18 families. In family members with combined defects, 72% (13/18) had had thrombosis as compared with 19% (4/21) of those with only protein S deficiency and 19% (4/21) of those with only the factor V mutation. In conclusion, APC resistance was found to be highly prevalent in thrombosis-prone families with protein S deficiency and was an additional genetic risk factor for thrombosis in these families. The results suggest thrombosis-prone families with protein S deficiency often to be affected by yet another genetic defect.

Evaluation of the relationship between protein S and C4b-binding protein isoforms in hereditary protein S deficiency demonstrating type I and type III deficiencies to be phenotypic variants of the same genetic disease.

Type III protein S deficiency is characterized by a low plasma level of free protein S, whereas the total concentration of protein S is normal. In contrast, both free and total protein S levels are low in type I deficiency. To elucidate the molecular mechanism behind the selective deficiency of free protein S in type III deficiency, the relationship between the plasma concentrations of beta-chain containing isoforms of C4b-binding protein (C4BP beta+) and different forms of protein S (free, bound, and total) was evaluated in 327 members of 18 protein S-deficient families. In normal relatives (n = 190), protein S correlated well with C4BP beta+, with free protein S (96 +/- 23 nmol/L) being equal to the molar excess of protein S (355 +/- 65 nmol/L) over C4BP beta+ (275 +/- 47 nmol/L). In protein S-deficient family members (n = 117), the equimolar relationship between protein S (215 +/- 50 nmol/L) and C4BP beta+ (228 +/- 51 nmol/L), together with the high affinity of the interaction, resulted in low levels of free protein S (16 +/- 10 nmol/L). Free protein S levels were distinctly low in protein S-deficient members, whereas in 47 of the protein S-deficient individuals, the concentration of total protein S was within the normal range, which fulfils the criteria for type III deficiency. The remaining 70 had low levels of both total and free protein S and, accordingly, would be type I deficient. Coexistence of type I and type III deficiency was found in 14 families, suggesting the two types of protein S deficiency to be phenotypic variants of the same genetic disease. Interestingly, not only protein S but also C4BP beta+ levels were decreased in orally anticoagulated controls and even more so in anticoagulated protein S-deficient members, suggesting that the concentration of C4BP beta+ is influenced by that of protein S. In conclusion, our results indicate that type I and type III deficiencies are phenotypic variants of the same genetic disease and that the low plasma concentrations of free protein S in both types are the result of an equimolar relationship between protein S and C4BP beta+.